Rabies glycoprotein virus-like particles (vlps)

ABSTRACT

The present invention is generally related to virus-like particles (VLPs) comprising rabies virus (RV) glycoproteins (G proteins) and methods for making and using them, including immunogenic compositions such as vaccines for the treatment and/or prevention of rabies virus infection.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. serial no. U.S. Ser. No.15/638,955, filed Jun. 30, 2017, which is a continuation of U.S. Ser.No. 13/883,745, filed Feb. 28, 2014 (now U.S. Pat. No. 9,724,405, issuedAug. 8, 2017), which is the U.S. national stage application ofInternational Application No. PCT/US2011/059602, which was filed on Nov.7, 2011 and claims priority to U.S. Provisional Application No.61/410,767, filed Nov. 5, 2010, the disclosures of each of which arehereby incorporated by reference in their entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:NOVV_047_03US_SeqList.txt, date recorded: Aug. 20, 2018, file size 7kilobytes).

TECHNICAL FIELD

The present invention is generally related to virus-like particles(VLPs) comprising rabies virus (RV) glycoproteins (G proteins) andmethods for making and using them, including immunogenic compositionssuch as vaccines for the treatment and/or prevention of rabies virusinfection.

BACKGROUND OF THE INVENTION

Rabies virus (RV) is a non-segmented negative-stranded RNA virus of theRhabdoviridae family and induces a fatal neurological disease in humansand animals. More than 70,000 human fatalities are reported annually andmillions of others require post-exposure treatment. Although significantadvances have been made in rabies prevention and control, the diseaseremains a major threat to public health and continues to cause numeroushuman deaths around the world. Canines remain the most importantreservoir in Asia, Africa and Latin America where most human rabiescases occur. In the developed countries, human rabies has declinedsignificantly during the past 50 years, primarily as a result of routinevaccination of pet animals. However, rabies transmission via exposure towild-life has emerged as a major cause of the disease. In the UnitedStates, more than 90% of animal rabies cases have been reported inwildlife, representing continual public health threats. Most human casesin the past decade have been associated with RV found in bats,particularly silver-haired bats.

Rhabdoviruses have two major structural components: a helicalribonucleoprotein core (RNP) and a surrounding envelope. The rabiesgenome encodes five proteins: nucleoprotein (N), phosphoprotein (P),matrix protein (M), glycoprotein (G) and polymerase (large protein) (L).The order of the genes in the wild-type rabies genome is3′-N-P-M-G-L-5′. The N, L and P proteins are associated with the coreRNP complex. The RNP complex consists of the RNA genome encapsidated bythe N in combination with polymerase L and the P protein. This complexserves as a template for virus transcription and replication. The viralenvelope component of RV is composed of a transmembrane glycoprotein (G)and a matrix (M) protein. The glycoprotein forms approximately 400trimeric spikes which are tightly arranged on the surface of the virus.The M protein is associated both with the envelope and the RNP and maybe the central protein of rhabdovirus assembly.

As noted above, rabies remains a major public health threat around theworld. Controlling rabies and protecting humans from rabies requiresseveral control strategies, such as routine immunization of pet animalsand wildlife carriers, pre-exposure immunization of people at risk, andpost-exposure treatment of people bitten by rabid animals. Althoughinactivated rabies virus (RV) vaccines prepared from cell culture aresafe and well-tolerated, they have multiple disadvantages. They aredifficult to manufacture, difficult to store, have low immunogenicity,and require multiple injections. Moreover, they are expensive and thusbeyond the reach of most people who need the vaccines in the developingcountries. In addition, these inactivated vaccines typically includeadjuvants which may cause unwanted side effects. Thus, safer, cheaper,and more efficacious RV vaccines are needed.

The present application addresses this need through the development of anovel method for the production of virus-like particles (VLPs)comprising the rabies glycoprotein (G).

SUMMARY OF THE INVENTION

The present invention relates to rabies virus (RV) virus-like particles(VLPs) for use in vaccines for the treatment and prevention of rabiesvirus infection. The RV VLPs of the invention have the potential toinduce potent immune responses in mammalian subjects against the rabiesvirus.

In a first aspect, the present invention provides RV VLPs comprising oneor more RV glycoproteins (G proteins). The RV G proteins may be derivedfrom any suitable RV strain, including, but not limited to, human,canine, bat, raccoon, skunk, and fox strains of RV. In one embodiment,the RV VLPs comprising one or more RV G proteins may be in the form ofmicelles. In some embodiments, the RV VLPs may comprise one or moreadditional RV proteins, selected from the nucleoprotein (N),phosphoprotein (P), matrix protein (M), and polymerase (large protein)(L). In a specific embodiment, the RV VLPs of the present invention maycomprise the RV matrix protein (M). In one embodiment, the M protein isderived from a human strain of RV. In another embodiment, the M proteinis derived from a canine strain of RV. In yet another embodiment, the Mprotein is derived from a bat strain of RV. In other embodiments, thematrix protein may be an M1 protein from an influenza virus strain. Inone embodiment, the influenza virus strain is an avian influenza virusstrain. In other embodiments, the M protein may be derived from aNewcastle Disease Virus (NDV) strain.

In one embodiment, the coding sequence of the RV G protein is furtheroptimized to enhance its expression in a suitable host cell. In oneembodiment, the host cell is an insect cell. In an exemplary embodiment,the insect cell is an Sf9 cell.

The RV VLPs of the present invention may be used for the preventionand/or treatment of RV infection. Thus, in another aspect, the inventionprovides a method for eliciting an immune response against RV. Themethod involves administering an immunologically effective amount of acomposition containing a RV VLP to a subject, such as a human or animalsubject.

In another aspect, the present invention provides pharmaceuticallyacceptable vaccine compositions comprising an RV VLP which comprises oneor more RV glycoproteins (G proteins).

In one embodiment, the invention comprises an immunogenic formulationcomprising at least one effective dose of an RV VLP which comprises oneor more RV glycoproteins (G proteins). In another embodiment, theinvention provides for a pharmaceutical pack or kit comprising one ormore containers filled with one or more of the ingredients of thevaccine formulations of the invention.

In another embodiment, the invention provides a method of formulating avaccine or antigenic composition that induces immunity to an infectionor at least one disease symptom thereof to a mammal, comprising addingto the formulation an effective dose of an RV VLP which comprises one ormore RV glycoproteins (G proteins). In a preferred embodiment, theinfection is an RV infection.

The RV VLPs of the invention are useful for preparing compositions thatstimulate an immune response that confers immunity or substantialimmunity to infectious agents. Thus, in one embodiment, the inventionprovides a method of inducing immunity to infections or at least onedisease symptom thereof in a subject, comprising administering at leastone effective dose of an RV VLP which comprises one or more RVglycoproteins (G proteins).

In yet another aspect, the invention provides a method of inducingsubstantial immunity to RV infection or at least one disease symptom ina subject, comprising administering at least one effective dose of an RVVLP which comprises one or more RV glycoproteins (G proteins).

Compositions of the invention can induce substantial immunity in avertebrate (e.g. a human or a canine) when administered to thevertebrate. Thus, in one embodiment, the invention provides a method ofinducing substantial immunity to RV infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of an RV VLP which comprises one or more RV glycoproteins (Gproteins). In another embodiment, the invention provides a method ofvaccinating a mammal against RV comprising administering to the mammal aprotection-inducing amount of an RV VLP which comprises one or more RVglycoproteins (G proteins). The prophylactic vaccine formulation issystemically administered, e.g., by subcutaneous or intramuscularinjection using a needle and syringe, or a needle-less injection device.In an exemplary embodiment, the vaccine formulation is administeredintramuscularly.

In another embodiment, the invention comprises a method of inducing aprotective antibody response to an infection or at least one symptomthereof in a subject, comprising administering at least one effectivedose of an RV VLP which comprises one or more RV glycoproteins (Gproteins).

In another embodiment, the invention comprises a method of inducing aprotective cellular response to RV infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of an RV VLP which comprises one or more RV glycoproteins (Gproteins).

In yet another aspect, the invention provides an isolated nucleic acidencoding a rabies glycoprotein (G protein). In an exemplary embodiment,the isolated nucleic acid encoding a rabies glycoprotein (G protein)protein is SEQ ID NO: 1.

In yet another aspect, the invention provides an isolated cellcomprising a nucleic acid encoding a rabies glycoprotein (G protein). Inan exemplary embodiment, the isolated nucleic acid encoding a rabiesglycoprotein (G protein) protein is SEQ ID NO: 1.

In yet another aspect, the invention provides a vector comprising anucleic acid encoding a rabies glycoprotein (G protein). In an exemplaryembodiment, the isolated nucleic acid encoding a rabies glycoprotein (Gprotein) protein is SEQ ID NO: 1. In one embodiment, the vector is abaculovirus vector.

In yet another aspect, the invention provides a method of making a RVVLP comprising one or more rabies glycoproteins (G proteins), comprising(a) transforming a host cell to express a nucleic acid encoding a rabiesglycoprotein (G protein); and (b) culturing said host cell underconditions conducive to the production of said RV VLPs. In oneembodiment, the nucleic acid encoding a rabies glycoprotein (G protein)is SEQ ID NO: 1. In another embodiment, the host cell is an insect cell.In a further embodiment, the host cell is an is an insect celltransfected with a baculovirus vector comprising a rabies glycoprotein(G protein).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the plasmid map for the pFastBac1 vector comprising therabies virus G nucleic acid sequence (SEQ ID NO: 1).

FIG. 2 depicts the results of western blotting for RV G proteins usinganti-RV rabbit sera under both reducing (FIG. 2A) and non-reducingconditions (FIG. 2B).

FIG. 3 depicts images of purified recombinant RV G protein particles inthe forms of micelles taken using negative stain electron microscopy ata magnification of 150,000×.

FIG. 4 depicts the results of antibody-induction assays in rabbitsadministered increasing dilutions of RV G particles.

FIG. 5 shows the protein sequence for the pFastBac1 vector comprisingthe rabies virus G nucliec acid sequence (SEQ ID NO: 1) (top) and the RVG protein sequence (SEQ ID NO: 2) (bottom).

FIG. 6 is a graph showing the anti-rabies virus antibody titers atdifferent days, plotted as the geometric mean for each immunizationregimen (n=5 for each immunization group).

DETAILED DESCRIPTION Definitions

As used herein the term “adjuvant” refers to a compound that, when usedin combination with a specific immunogen (e.g. an RV VLP which comprisesone or more RV glycoproteins (G proteins)) in a formulation, willaugment or otherwise alter or modify the resultant immune response.Modification of the immune response includes intensification orbroadening the specificity of either or both antibody and cellularimmune responses. Modification of the immune response can also meandecreasing or suppressing certain antigen-specific immune responses.

As use herein, the term “antigenic formulation” or “antigeniccomposition” refers to a preparation which, when administered to avertebrate, especially a bird or a mammal, will induce an immuneresponse.

As used herein the term “avian influenza virus” refers to influenzaviruses found chiefly in birds but that can also infect humans or otheranimals. In some instances, avian influenza viruses may be transmittedor spread from one human to another. An avian influenza virus thatinfects humans has the potential to cause an influenza pandemic, i.e.,morbidity and/or mortality in humans. A pandemic occurs when a newstrain of influenza virus (a virus in which human have no naturalimmunity) emerges, spreading beyond individual localities, possiblyaround the globe, and infecting many humans at once.

As used herein an “effective dose” generally refers to that amount of anRV VLP which comprises one or more RV glycoproteins (G proteins)sufficient to induce immunity, to prevent and/or ameliorate an infectionor to reduce at least one symptom of an infection or disease, and/or toenhance the efficacy of another dose of an RV VLP which comprises one ormore RV glycoproteins (G proteins). An effective dose may refer to theamount of an RV VLP which comprises one or more RV glycoproteins (Gproteins) sufficient to delay or minimize the onset of an infection ordisease. An effective dose may also refer to the amount of an RV VLPwhich comprises one or more RV glycoproteins (G proteins) that providesa therapeutic benefit in the treatment or management of an infection ordisease. Further, an effective dose is the amount with respect to an RVVLP which comprises one or more RV glycoproteins (G proteins) alone, orin combination with other therapies, that provides a therapeutic benefitin the treatment or management of an infection or disease. An effectivedose may also be the amount sufficient to enhance a subject's (e.g., ahuman's) own immune response against a subsequent exposure to aninfectious agent or disease. Levels of immunity can be monitored, e.g.,by measuring amounts of neutralizing secretory and/or serum antibodies,e.g., by plaque neutralization, complement fixation, enzyme-linkedimmunosorbent, or microneutralization assay, or by measuring cellularresponses, such as, but not limited to cytotoxic T cells, antigenpresenting cells, helper T cells, dendritic cells and/or other cellularresponses. T cell responses can be monitored, e.g., by measuring, forexample, the amount of CD4⁺ and CD8⁺ cells present using specificmarkers by fluorescent flow cytometry or T cell assays, such as but notlimited to T-cell proliferation assay, T-cell cytotoxic assay, TETRAMERassay, and/or ELISPOT assay. In the case of a vaccine, an “effectivedose” is one that prevents disease and/or reduces the severity ofsymptoms.

As used herein, the term “effective amount” refers to an amount of an RVVLP which comprises one or more RV glycoproteins (G proteins) necessaryor sufficient to realize a desired biologic effect. An effective amountof the composition would be the amount that achieves a selected result,and such an amount could be determined as a matter of routineexperimentation by a person skilled in the art. For example, aneffective amount for preventing, treating and/or ameliorating aninfection could be that amount necessary to cause activation of theimmune system, resulting in the development of an antigen specificimmune response upon exposure to an RV VLP which comprises one or moreRV glycoproteins (G proteins). The term is also synonymous with“sufficient amount.” In another embodiment, the effective amount is theamount by weight of a RV G micelle that enduces seroprotection in arelevant animal model, animal or human patient in a desired number ofdays, e.g. 7, 10, 14 or more days.

As used herein, the term “expression” refers to the process by whichpolynucleic acids are transcribed into mRNA and translated intopeptides, polypeptides, or proteins. If the polynucleic acid is derivedfrom genomic DNA, expression may, if an appropriate eukaryotic host cellor organism is selected, include splicing of the mRNA. In the context ofthe present invention, the term also encompasses the yield of RV G genemRNA and RV G proteins achieved following expression thereof.

As used herein, the term “G protein” or “G glycoprotein” or “G proteinpolypeptide” refers to a polypeptide or protein having all or part of anamino acid sequence of an RV G protein polypeptide.

As used herein, the terms “immunogens” or “antigens” refer to substancessuch as proteins, peptides, peptides, nucleic acids that are capable ofeliciting an immune response. Both terms also encompass epitopes, andare used interchangeably.

As used herein the term “immune stimulator” refers to a compound thatenhances an immune response via the body's own chemical messengers(cytokines). These molecules comprise various cytokines, lymphokines andchemokines with immunostimulatory, immunopotentiating, andpro-inflammatory activities, such as interferons (IFN-γ), interleukins(e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g.,granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and otherimmunostimulatory molecules, such as macrophage inflammatory factor,Flt3 ligand, B7.1; B7.2, etc. The immune stimulator molecules can beadministered in the same formulation as VLPs of the invention, or can beadministered separately. Either the protein or an expression vectorencoding the protein can be administered to produce an immunostimulatoryeffect.

As use herein, the term “immunogenic formulation” refers to apreparation which, when administered to a vertebrate, e.g. a mammal,will induce an immune response.

As use herein, the term “infectious agent” refers to microorganisms thatcause an infection in a vertebrate. Usually, the organisms are viruses,bacteria, parasites, protozoa and/or fungi.

As used herein, the term “multivalent” refers to compositions which haveone or more antigenic proteins/peptides or immunogens against multipletypes or strains of infectious agents or diseases, e.g. more than one RVG protein type, strain, sequence, etc.

As used herein, the term “pharmaceutically acceptable vaccine” refers toa formulation which contains an RV VLP which comprises one or more RVglycoproteins (G proteins), which is in a form that is capable of beingadministered to a vertebrate and which induces a protective immuneresponse sufficient to induce immunity to prevent and/or ameliorate aninfection or disease, and/or to reduce at least one symptom of aninfection or disease, and/or to enhance the efficacy of another dose ofan RV VLP which comprises one or more RV glycoproteins (G proteins).Typically, the vaccine comprises a conventional saline or bufferedaqueous solution medium in which the composition of the presentinvention is suspended or dissolved. In this form, the composition ofthe present invention can be used conveniently to prevent, ameliorate,or otherwise treat an infection. Upon introduction into a host, thevaccine is able to provoke an immune response including, but not limitedto, the production of antibodies and/or cytokines and/or the activationof cytotoxic T cells, antigen presenting cells, helper T cells,dendritic cells and/or other cellular responses.

As used herein, the phrase “protective immune response” or “protectiveresponse” refers to an immune response mediated by antibodies against aninfectious agent or disease, which is exhibited by a vertebrate (e.g., ahuman), that prevents or ameliorates an infection or reduces at leastone disease symptom thereof. An RV VLP of the present invention whichcomprises one or more RV glycoproteins (G proteins) can stimulate theproduction of antibodies that, for example, neutralize infectiousagents, blocks infectious agents from entering cells, blocks replicationof the infectious agents, and/or protect host cells from infection anddestruction. The term can also refer to an immune response that ismediated by T-lymphocytes and/or other white blood cells against aninfectious agent or disease, exhibited by a vertebrate (e.g., a human),that prevents or ameliorates infection or disease, or reduces at leastone symptom thereof.

As use herein, the term “vertebrate” or “subject” or “patient” refers toany member of the subphylum cordata, including, without limitation,humans and other primates, including non-human primates such aschimpanzees and other apes and monkey species. Farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats (includingcotton rats) and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like are also non-limiting examples. The terms “mammals”and “animals” are included in this definition. Both adult and newbornindividuals are intended to be covered. In particular, humans, domesticmammals, and farm animals are appropriate recipients of an RV vaccine ortherapeutic.

As used herein, the term “virus-like particle” (VLP) refers to astructure that in at least one attribute resembles a virus but which hasnot been demonstrated to be infectious. Virus-like particles inaccordance with the invention do not carry genetic information encodingfor the proteins of the virus-like particles. In general, virus-likeparticles lack a viral genome and, therefore, are noninfectious. Inaddition, virus-like particles can often be produced in large quantitiesby heterologous expression and can be easily purified.

As used herein, the term “chimeric VLP” refers to VLPs that containproteins, or portions thereof, from at least two different infectiousagents (heterologous proteins). Usually, one of the proteins is derivedfrom a virus that can drive the formation of VLPs from host cells.Examples, for illustrative purposes, are the avian influenza M proteinand/or the RV G protein. The terms RV VLPs and chimeric VLPs can be usedinterchangeably where appropriate.

As used herein, the term “vaccine” refers to a preparation of dead orweakened pathogens, or of derived antigenic determinants that is used toinduce formation of antibodies or immunity against the pathogen. Avaccine is given to provide immunity to the disease, for example,influenza, which is caused by influenza viruses. In addition, the term“vaccine” also refers to a suspension or solution of an immunogen (e.g.an RV VLP which comprises one or more RV glycoproteins (G proteins))that is administered to a vertebrate to produce protective immunity,i.e., immunity that prevents or reduces the severity of diseaseassociated with infection. The present invention provides for vaccinecompositions that are immunogenic and may provide protection against adisease associated with infection.

Rabies Virus (RV) Virus-Like Particles (VLPs)

In one aspect, the invention relates RV virus-like particles (VLPs)comprising one or more RV glycoproteins (G proteins) that can beformulated into vaccines or antigenic formulations for protectingvertebrates (e.g. humans and domestic animals) against RV infection orat least one disease symptom thereof. In some embodiments, the VLPcomprising one or more RV glycoproteins (G proteins) further comprisesadditional RV proteins, such as N, P, M, and L. In other embodiments,the VLP comprising one or more RV glycoproteins (G proteins) furthercomprises proteins from heterologous strains of virus, such as influenzavirus proteins HA, NA, and M1. In one embodiment, the influenza virusprotein M1 is derived from an avian influenza virus strain (see U.S.application Ser. No. 13/280,043, which is incorporated herein byreference in its entirety).

RV Vaccines

Since RV infection can be prevented by providing neutralizing antibodiesto a vertebrate, a vaccine comprising an RV VLP which comprises one ormore RV glycoproteins (G proteins) may induce, when administered to avertebrate, neutralizing antibodies in vivo. The RV VLPs which compriseone or more RV glycoproteins (G proteins) are favorably used for theprevention and/or treatment of RV infection. Thus, another aspect ofthis disclosure concerns a method for eliciting an immune responseagainst RV. The method involves administering an immunologicallyeffective amount of a composition containing an RV VLP which comprisesone or more RV glycoproteins (G proteins) to a subject (such as a humanor animal subject). Administration of an immunologically effectiveamount of the composition elicits an immune response specific forepitopes present on the RV G protein. Such an immune response caninclude B cell responses (e.g., the production of neutralizingantibodies) and/or T cell responses (e.g., the production of cytokines).Preferably, the immune response elicited by the RV G protein includeselements that are specific for at least one conformational epitopepresent on the RV G protein. In one embodiment, the immune response isspecific for an epitope present on an RV G protein found in the micelleconformation. The RV G proteins and compositions can be administered toa subject without enhancing viral disease following contact with RV.Preferably, the RV G proteins disclosed herein and suitably formulatedimmunogenic compositions elicit a Th1 biased immune response thatreduces or prevents infection with a RV and/or reduces or prevents apathological response following infection with a RV.

In one embodiment, the RV G proteins of the present invention are foundin the form of micelles (e.g. rosettes). See example 2. In oneembodiment, the micelles are purified following expression in a hostcell. When administered to a subject, the micelles of the presentinvention preferably induce neutralizing antibodies. In someembodiments, the micelles may be administered with an adjuvant. In otherembodiments, the micelles may be administered without an adjuvant.

In another embodiment, the invention encompasses RV virus-like particles(VLPs) comprising a RV G protein that can be formulated into vaccines orantigenic formulations for protecting vertebrates (e.g. humans) againstRV infection or at least one disease symptom thereof. The presentinvention also relates to RV VLPs and vectors comprising wild-type andmutated RV genes or a combination thereof derived from different strainsof RV virus, which when transfected into host cells, will produce viruslike particles (VLPs) comprising RV proteins.

In some embodiments, RV virus-like particles may further comprise atleast one viral matrix protein (e.g. an RV M protein). In oneembodiment, the M protein is derived from a human strain of RV. Inanother embodiment, the M protein is derived from an alternative strainof RV, such as a canine, bat, raccoon, or skunk strain of RV. In otherembodiments, the matrix protein may be an M1 protein from a strain ofinfluenza virus. In one embodiment, the strain of influenza virus is anavian influenza strain. In an exemplary embodiment, the avian influenzastrain is the H5N1 strain A/Indonesia/5/05. In other embodiments, thematrix protein may be from Newcastle Disease Virus (NDV).

In further embodiments, VLPs of the invention may comprise one or moreheterologous immunogens, such as influenza hemagglutinin (HA) and/orneuraminidase (NA).

In some embodiments, the invention also comprises combinations ofdifferent RV G, N, P, M, and L proteins from the same and/or differentstrains in one or more VLPs. In addition, the VLPs can include one ormore additional molecules for the enhancement of an immune response.

In another embodiment of the invention, the RV VLPs can carry agentssuch as nucleic acids, siRNA, microRNA, chemotherapeutic agents, imagingagents, and/or other agents that need to be delivered to a patient.

VLPs of the invention are useful for preparing vaccines and immunogeniccompositions. One important feature of VLPs is the ability to expresssurface proteins of interest so that the immune system of a vertebrateinduces an immune response against the protein of interest. However, notall proteins can be expressed on the surface of VLPs. There may be manyreasons why certain proteins are not expressed, or be poorly expressed,on the surface of VLPs. One reason is that the protein is not directedto the membrane of a host cell or that the protein does not have atransmembrane domain. As an example, sequences near the carboxylterminus of influenza hemagglutinin may be important for incorporationof HA into the lipid bilayer of the mature influenza envelopednucleocapsids and for the assembly of HA trimer interaction with theinfluenza matrix protein M1 (Ali, et al., (2000) J. Virol. 74, 8709-19).

Thus, one embodiment of the invention comprises chimeric VLPs comprisinga G protein from RV and at least one immunogen which is not normallyefficiently expressed on the cell surface or is not a normal RV protein.In one embodiment, the RV G protein may be fused with an immunogen ofinterest. In another embodiment, the RV G protein associates with theimmunogen via the transmembrane domain and cytoplasmic tail of aheterologous viral surface membrane protein, e.g., MMTV envelopeprotein.

Other chimeric VLPs of the invention comprise VLPs comprising a RV Gprotein and at least one protein from a heterologous infectious agent.Examples of heterologous infectious agent include but are not limited toa virus, a bacterium, a protozoan, a fungi and/or a parasite. In oneembodiment, the immunogen from another infectious agent is aheterologous viral protein. In another embodiment, the protein from aheterologous infectious agent is an envelope-associated protein. Inanother embodiment, the protein from another heterologous infectiousagent is expressed on the surface of VLPs. In another embodiment, theprotein from an infectious agent comprises an epitope that will generatea protective immune response in a vertebrate. In one embodiment, theprotein from another infectious agent is co-expressed with a RV Gprotein. In another embodiment, the protein from another infectiousagent is fused to a RV G protein. In another embodiment, only a portionof a protein from another infectious agent is fused to a RV G protein.In another embodiment, only a portion of a protein from anotherinfectious agent is fused to a portion of a RV G protein. In anotherembodiment, the portion of the protein from another infectious agentfused to a RV G protein is expressed on the surface of VLPs.

The invention also encompasses variants of the proteins expressed on orin the VLPs of the invention. The variants may contain alterations inthe amino acid sequences of the constituent proteins. The term “variant”with respect to a protein refers to an amino acid sequence that isaltered by one or more amino acids with respect to a reference sequence.The variant can have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties, e.g., replacement ofleucine with isoleucine. Alternatively, a variant can have“nonconservative” changes, e.g., replacement of a glycine with atryptophan. Analogous minor variations can also include amino aciddeletion or insertion, or both. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without eliminatingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software.

Natural variants can occur due to mutations in the proteins. Thesemutations may lead to antigenic variability within individual groups ofinfectious agents, for example influenza. Thus, a person infected with,for example, an influenza strain develops antibody against that virus,as newer virus strains appear, the antibodies against the older strainsno longer recognize the newer virus and re-infection can occur. Theinvention encompasses all antigenic and genetic variability of proteinsfrom infectious agents for making VLPs.

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (“Ausubel”). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thecloning and mutating of RV G molecules, etc. Thus, the invention alsoencompasses using known methods of protein engineering and recombinantDNA technology to improve or alter the characteristics of the proteinsexpressed on or in the VLPs of the invention. Various types ofmutagenesis can be used to produce and/or isolate variant nucleic acidsthat encode for protein molecules and/or to further modify/mutate theproteins in or on the VLPs of the invention. They include but are notlimited to site-directed, random point mutagenesis, homologousrecombination (DNA shuffling), mutagenesis using uracil containingtemplates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

The invention further comprises protein variants which show substantialbiological activity, e.g., able to elicit an effective antibody responsewhen expressed on or in VLPs of the invention. Such variants includedeletions, insertions, inversions, repeats, and substitutions selectedaccording to general rules known in the art so as have little effect onactivity.

Methods of cloning the proteins are known in the art. For example, thegene encoding a specific RV protein can be isolated by RT-PCR frompolyadenylated mRNA extracted from cells which had been infected withrabies virus. The resulting product gene can be cloned as a DNA insertinto a vector. The term “vector” refers to the means by which a nucleicacid can be propagated and/or transferred between organisms, cells, orcellular components. Vectors include plasmids, viruses, bacteriophages,pro-viruses, phagemids, transposons, artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that is not autonomously replicating. In many, but not all, commonembodiments, the vectors of the present invention are plasmids orbacmids.

Thus, the invention comprises nucleotides that encode proteins,including chimeric molecules, cloned into an expression vector that canbe expressed in a cell that induces the formation of VLPs of theinvention. An “expression vector” is a vector, such as a plasmid that iscapable of promoting expression, as well as replication of a nucleicacid incorporated therein. Typically, the nucleic acid to be expressedis “operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer. In oneembodiment, the nucleotides encode for a RV G protein (as discussedabove). In another embodiment, the vector further comprises nucleotidesthat encode the RV M protein. In another embodiment, the vector furthercomprises nucleotides that encode the M and/or N RV proteins. In anotherembodiment, the vector further comprises nucleotides that encode the M,L and/or N RV proteins. In an exemplary embodiment, the expressionvector is a baculovirus vector.

In some embodiments of the invention, proteins may comprise mutationscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedprotein or how the proteins are made. Nucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the human mRNA to thosepreferred by insect cells such as Sf9 cells. See U.S. Patent Publication2005/0118191, herein incorporated by reference in its entirety for allpurposes.

In addition, the nucleotides can be sequenced to ensure that the correctcoding regions were cloned and do not contain any unwanted mutations.The nucleotides can be subcloned into an expression vector (e.g.baculovirus) for expression in any cell. The above is only one exampleof how the RV viral proteins can be cloned. A person with skill in theart understands that additional methods are available and are possible.

The invention also provides for constructs and/or vectors that compriseRV nucleotides that encode for RV structural genes, including G, M, N,L, P, or portions thereof, and/or any chimeric molecule described above.The vector may be, for example, a phage, plasmid, viral, or retroviralvector. The constructs and/or vectors that comprise RV structural genes,including G, M, N, L, P, or portions thereof, and/or any chimericmolecule described above, should be operatively linked to an appropriatepromoter, such as the AcMNPV polyhedrin promoter (or other baculovirus),phage lambda PL promoter, the E. coli lac, phoA and tac promoters, theSV40 early and late promoters, and promoters of retroviral LTRs arenon-limiting examples. Other suitable promoters will be known to theskilled artisan depending on the host cell and/or the rate of expressiondesired. The expression constructs will further contain sites fortranscription initiation, termination, and, in the transcribed region, aribosome-binding site for translation. The coding portion of thetranscripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

Expression vectors will preferably include at least one selectablemarker. Such markers include dihydrofolate reductase, G418 or neomycinresistance for eukaryotic cell culture and tetracycline, kanamycin orampicillin resistance genes for culturing in E. coli and other bacteria.Among vectors preferred are virus vectors, such as baculovirus, poxvirus(e.g., vaccinia virus, avipox virus, canarypox virus, fowlpox virus,raccoonpox virus, swinepox virus, etc.), adenovirus (e.g., canineadenovirus), herpesvirus, and retrovirus. Other vectors that can be usedwith the invention comprise vectors for use in bacteria, which comprisepQE70, pQE60 and pQE-9, pBluescript vectors, Phagescript vectors, pNH8A,pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5.Among preferred eukaryotic vectors are pFastBac1 pWINEO, pSV2CAT, pOG44,pXT1 and pSG, pSVK3, pBPV, pMSG, and pSVL. Other suitable vectors willbe readily apparent to the skilled artisan. In one embodiment, thevector that comprises nucleotides encoding for RV genes, including RV Ggenes, as well as genes for M, N, L, P, or portions thereof, and/or anychimeric molecule described above, is pFastBac.

The recombinant constructs mentioned above could be used to transfect,infect, or transform and can express RV proteins, including a RV Gprotein and at least one immunogen. In one embodiment, the recombinantconstruct comprises a RV G, M, N, L, P, or portions thereof, and/or anymolecule described above, into eukaryotic cells and/or prokaryoticcells. Thus, the invention provides for host cells which comprise avector (or vectors) that contain nucleic acids which code for RVstructural genes, including a RV G; and at least one immunogen such asbut not limited to RV N, L, and P, or portions thereof, and/or anymolecule described above, and permit the expression of genes, includingRV G, N, L, or P or portions thereof, and/or any molecule describedabove in the host cell under conditions which allow the formation ofVLPs.

Among eukaryotic host cells are yeast, insect, avian, plant, C. elegans(or nematode) and mammalian host cells. Non limiting examples of insectcells are, Spodoptera frugiperda (Sf) cells, e.g. Sf9, Sf21,Trichoplusia ni cells, e.g. High Five cells, and Drosophila S2 cells.Examples of fungi (including yeast) host cells are S. cerevisiae,Kluyveromyces lactis (K. lactis), species of Candida including C.albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomycespombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica. Examples ofmammalian cells are COS cells, baby hamster kidney cells, mouse L cells,LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney(HEK) cells, and African green monkey cells, CV1 cells, HeLa cells, MDCKcells, Vero and Hep-2 cells. Xenopus laevis oocytes, or other cells ofamphibian origin, may also be used. Examples of prokaryotic host cellsinclude bacterial cells, for example, E. coli, B. subtilis, Salmonellatyphi and mycobacteria.

Vectors, e.g., vectors comprising polynucleotides of a RV G protein; andat least one immunogen including but not limited to RV N, L, P, orportions thereof, and/or any chimeric molecule described above, can betransfected into host cells according to methods well known in the art.For example, introducing nucleic acids into eukaryotic cells can be bycalcium phosphate co-precipitation, electroporation, microinjection,lipofection, and transfection employing polyamine transfection reagents.In one embodiment, the vector is a recombinant baculovirus. In anotherembodiment, the recombinant baculovirus is transfected into a eukaryoticcell. In a preferred embodiment, the cell is an insect cell. In anotherembodiment, the insect cell is a Sf9 cell.

This invention also provides for constructs and methods that willincrease the efficiency of VLP production. For example, the addition ofleader sequences to the RV G, M, N, L, P, or portions thereof, and/orany chimeric or heterologous molecules described above, can improve theefficiency of protein transporting within the cell. For example, aheterologous signal sequence can be fused to the RV G, M, N, L, P, orportions thereof, and/or any chimeric or heterologous molecule describedabove. In one embodiment, the signal sequence can be derived from thegene of an insect cell and fused to the RV G, M, N, L, P, or portionsthereof, and/or any chimeric or heterologous molecules described above.In another embodiment, the signal peptide is the chitinase signalsequence, which works efficiently in baculovirus expression systems.

Another method to increase efficiency of VLP production is to codonoptimize the nucleotides that encode RV including a RV G protein, M, N,L, P, or portions thereof, and/or any chimeric or heterologous moleculesdescribed above for a specific cell type. In one embodiment, nucleicacids are codon optimized for expression in insect cells. In anexemplary embodiment, the insect cells are Sf9 insect cells.

The invention also provides for methods of producing VLPs, the methodscomprising expressing RV genes including a RV G protein under conditionsthat allow VLP formation. Depending on the expression system and hostcell selected, the VLPs are produced by growing host cells transformedby an expression vector under conditions whereby the recombinantproteins are expressed and VLPs are formed. In one embodiment, theinvention comprises a method of producing a VLP, comprising transfectingvectors encoding at least RV G protein into a suitable host cell andexpressing the RV G protein under conditions that allow VLP formation.In another embodiment, the eukaryotic cell is selected from the groupconsisting of, yeast, insect, amphibian, avian or mammalian cells. Theselection of the appropriate growth conditions is within the skill ofone of ordinary skill in the art.

Methods to grow cells engineered to produce VLPs of the inventioninclude, but are not limited to, batch, batch-fed, continuous andperfusion cell culture techniques. Cell culture means the growth andpropagation of cells in a bioreactor (a fermentation chamber) wherecells propagate and express protein (e.g. recombinant proteins) forpurification and isolation. Typically, cell culture is performed understerile, controlled temperature and atmospheric conditions in abioreactor. A bioreactor is a chamber used to culture cells in whichenvironmental conditions such as temperature, atmosphere, agitationand/or pH can be monitored. In one embodiment, the bioreactor is astainless steel chamber. In another embodiment, the bioreactor is apre-sterilized plastic bag (e.g. Cellbag®, Wave Biotech, Bridgewater,N.J.). In other embodiment, the pre-sterilized plastic bags are about 50L to 1000 L bags.

The VLPs are then isolated using methods that preserve the integritythereof, such as by gradient centrifugation, e.g., cesium chloride,sucrose and iodixanol, as well as standard purification techniquesincluding, e.g., ion exchange and gel filtration chromatography.

The following is an example of how VLPs of the invention can be made,isolated and purified. Usually VLPs are produced from recombinant celllines engineered to create VLPs when the cells are grown in cell culture(see above). A person of skill in the art would understand that thereare additional methods that can be utilized to make and purify VLPs ofthe invention, thus the invention is not limited to the methoddescribed.

Production of VLPs of the invention can start by seeding Sf9 cells(non-infected) into shaker flasks, allowing the cells to expand andscaling up as the cells grow and multiply (for example from a 125-mlflask to a 50 L Wave bag). The medium used to grow the cell isformulated for the appropriate cell line (preferably serum free media,e.g. insect medium ExCell-420, JRH). Next, the cells are infected withrecombinant baculovirus at the most efficient multiplicity of infection(e.g. from about 1 to about 3 plaque forming units per cell). Onceinfection has occurred, the RV G protein, and/or any chimeric orheterologous molecule described above, are expressed from the virusgenome, self assemble into VLPs and are secreted from the cellsapproximately 24 to 72 hours post infection. Usually, infection is mostefficient when the cells are in mid-log phase of growth (4-8×10⁶cells/ml) and are at least about 90% viable.

VLPs can be harvested approximately 48 to 96 hours post infection, whenthe levels of VLPs in the cell culture medium are near the maximum butbefore extensive cell lysis. The Sf9 cell density and viability at thetime of harvest can be about 0.5×10⁶ cells/ml to about 1.5×10⁶ cells/mlwith at least 20% viability, as shown by dye exclusion assay. Next, themedium is removed and clarified. NaCl can be added to the medium to aconcentration of about 0.4 to about 1.0 M, preferably to about 0.5 M, toavoid VLP aggregation. The removal of cell and cellular debris from thecell culture medium containing VLPs of the invention can be accomplishedby tangential flow filtration (TFF) with a single use, pre-sterilizedhollow fiber 0.5 or 1.00 μm filter cartridge or a similar device.

Next, VLPs in the clarified culture medium can be concentrated byultra-filtration using a disposable, pre-sterilized 500,000 molecularweight cut off hollow fiber cartridge. The concentrated VLPs can bediafiltrated against 10 volumes pH 7.0 to 8.0 phosphate-buffered saline(PBS) containing 0.5 M NaCl to remove residual medium components.

The concentrated, diafiltered VLPs can be furthered purified on a 20% to60% discontinuous sucrose gradient in pH 7.2 PBS buffer with 0.5 M NaClby centrifugation at 6,500×g for 18 hours at about 4° C. to about 10° C.Usually VLPs will form a distinctive visible band between about 30% toabout 40% sucrose or at the interface (in a 20% and 60% step gradient)that can be collected from the gradient and stored. This product can bediluted to comprise 200 mM of NaCl in preparation for the next step inthe purification process. This product contains VLPs and may containintact baculovirus particles.

Further purification of VLPs can be achieved by anion exchangechromatography, or 44% isopycnic sucrose cushion centrifugation. Inanion exchange chromatography, the sample from the sucrose gradient (seeabove) is loaded into column containing a medium with an anion (e.g.Matrix Fractogel EMD TMAE) and eluded via a salt gradient (from about0.2 M to about 1.0 M of NaCl) that can separate the VLP from othercontaminates (e.g. baculovirus and DNA/RNA). In the sucrose cushionmethod, the sample comprising the VLPs is added to a 44% sucrose cushionand centrifuged for about 18 hours at 30,000 g. VLPs form a band at thetop of 44% sucrose, while baculovirus precipitates at the bottom andother contaminating proteins stay in the 0% sucrose layer at the top.The VLP peak or band is collected.

The intact baculovirus can be inactivated, if desired. Inactivation canbe accomplished by chemical methods, for example, formalin orβ-propiolactone (BPL). Removal and/or inactivation of intact baculoviruscan also be largely accomplished by using selective precipitation andchromatographic methods known in the art, as exemplified above. Methodsof inactivation comprise incubating the sample containing the VLPs in0.2% of BPL for 3 hours at about 25° C. to about 27° C. The baculoviruscan also be inactivated by incubating the sample containing the VLPs at0.05% BPL at 4° C. for 3 days, then at 37° C. for one hour.

After the inactivation/removal step, the product comprising VLPs can berun through another diafiltration step to remove any reagent from theinactivation step and/or any residual sucrose, and to place the VLPsinto the desired buffer (e.g. PBS). The solution comprising VLPs can besterilized by methods known in the art (e.g. sterile filtration) andstored in the refrigerator or freezer.

The above techniques can be practiced across a variety of scales. Forexample, T-flasks, shake-flasks, spinner bottles, up to industrial sizedbioreactors. The bioreactors can comprise either a stainless steel tankor a pre-sterilized plastic bag (for example, the system sold by WaveBiotech, Bridgewater, N.J.). A person with skill in the art will knowwhat is most desirable for their purposes.

Expansion and production of baculovirus expression vectors and infectionof cells with recombinant baculovirus to produce recombinant RV VLPs canbe accomplished in insect cells, for example Sf9 insect cells aspreviously described. In one embodiment, the cells are Sf9 infected withrecombinant baculovirus engineered to produce RV VLPs.

Pharmaceutical or Vaccine Formulations and Administration

The pharmaceutical compositions useful herein contain a pharmaceuticallyacceptable carrier, including any suitable diluent or excipient, whichincludes any pharmaceutical agent that does not itself induce theproduction of an immune response harmful to the vertebrate receiving thecomposition, and which may be administered without undue toxicity and anRV VLP which comprises one or more RV glycoproteins (G proteins). Asused herein, the term “pharmaceutically acceptable” means being approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopia, European Pharmacopia or other generally recognizedpharmacopia for use in mammals, and more particularly in humans. Thesecompositions can be useful as a vaccine and/or antigenic compositionsfor inducing a protective immune response in a vertebrate.

The invention encompasses a pharmaceutically acceptable vaccinecomposition comprising an RV VLP which comprises one or more RVglycoproteins (G proteins). In one embodiment, the pharmaceuticallyacceptable vaccine composition comprises VLPs comprising at least one RVG protein and at least one additional immunogen. In another embodiment,the pharmaceutically acceptable vaccine composition comprises VLPscomprising at least one RV G protein and at least one RV M protein. Inanother embodiment, the pharmaceutically acceptable vaccine compositioncomprises VLPs comprising at least one RV G protein and at least oneinfluenza M protein. In another embodiment, the pharmaceuticallyacceptable vaccine composition comprises VLPs comprising at least one RVG protein and at least one avian influenza M1 protein.

The invention also encompasses a kit for immunizing a vertebrate, suchas a human subject, comprising VLPs that comprise at least one RV Gprotein.

In one embodiment, the invention comprises an immunogenic formulationcomprising at least one effective dose of an RV VLP which comprises oneor more RV glycoproteins (G proteins).

The immunogenic formulation of the invention comprises an RV VLP whichcomprises one or more RV glycoproteins (G proteins), and apharmaceutically acceptable carrier or excipient. Pharmaceuticallyacceptable carriers include but are not limited to saline, bufferedsaline, dextrose, water, glycerol, sterile isotonic aqueous buffer, andcombinations thereof. A thorough discussion of pharmaceuticallyacceptable carriers, diluents, and other excipients is presented inRemington's Pharmaceutical Sciences (Mack Pub. Co. N.J. currentedition). The formulation should suit the mode of administration. In apreferred embodiment, the formulation is suitable for administration tohumans, preferably is sterile, non-particulate and/or non-pyrogenic.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. The composition can be asolid form, such as a lyophilized powder suitable for reconstitution, aliquid solution, suspension, emulsion, tablet, pill, capsule, sustainedrelease formulation, or powder. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc.

The invention also provides for a pharmaceutical pack or kit comprisingone or more containers filled with one or more of the ingredients of thevaccine formulations of the invention. In one embodiment, the kitcomprises two containers, one containing an RV VLP which comprises oneor more RV glycoproteins (G proteins), and the other containing anadjuvant. Associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The invention also provides that the formulation be packaged in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of composition. In one embodiment, the composition issupplied as a liquid, in another embodiment, as a dry sterilizedlyophilized powder or water free concentrate in a hermetically sealedcontainer and can be reconstituted, e.g., with water or saline to theappropriate concentration for administration to a subject.

In an alternative embodiment, the composition is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the composition. Preferably, the liquid form of thecomposition is supplied in a hermetically sealed container at leastabout 50 μg/ml, more preferably at least about 100 μg/ml, at least about200 μg/ml, at least 500 μg/ml, or at least 1 mg/ml.

As an example, RV VLPs comprising one or more RV G proteins areadministered in an effective amount or quantity (as defined above)sufficient to stimulate an immune response, each a response against oneor more strains of RV. Administration of the RV VLP which comprises oneor more RV glycoproteins (G proteins) elicits immunity against RV.Typically, the dose can be adjusted within this range based on, e.g.,age, physical condition, body weight, sex, diet, time of administration,and other clinical factors. The prophylactic vaccine formulation issystemically administered, e.g., by subcutaneous or intramuscularinjection using a needle and syringe, or a needle-less injection device.In an exemplary embodiment, the vaccine formulation is administeredintramuscularly.

Thus, the invention also comprises a method of formulating a vaccine orantigenic composition that induces immunity to an infection or at leastone disease symptom thereof to a mammal, comprising adding to theformulation an effective dose of an RV VLP which comprises one or moreRV glycoproteins (G proteins). In one embodiment, the infection is an RVinfection.

While stimulation of immunity with a single dose is possible, additionaldosages can be administered, by the same or different route, to achievethe desired effect. In neonates and infants, for example, multipleadministrations may be required to elicit sufficient levels of immunity.Administration can continue at intervals throughout childhood, asnecessary to maintain sufficient levels of protection againstinfections, e.g. RV infection. Similarly, adults who are particularlysusceptible to repeated or serious infections, such as, for example,health care workers, day care workers, family members of young children,the elderly, and individuals with compromised cardiopulmonary functionmay require multiple immunizations to establish and/or maintainprotective immune responses. Levels of induced immunity can bemonitored, for example, by measuring amounts of neutralizing secretoryand serum antibodies, and dosages adjusted or vaccinations repeated asnecessary to elicit and maintain desired levels of protection.

Methods of administering a composition comprising an RV VLP whichcomprises one or more RV glycoproteins (G proteins) (e.g. vaccine and/orantigenic formulations) include, but are not limited to, parenteraladministration (e.g., intradermal, intramuscular, intravenous andsubcutaneous), epidural, and mucosal (e.g., intranasal and oral orpulmonary routes or by suppositories). In a specific embodiment,compositions of the present invention are administered intramuscularly,intravenously, subcutaneously, transdermally or intradermally. Thecompositions may be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucous, colon, conjunctiva,nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents.

Vaccines and/or immunogenic formulations of the invention may also beadministered on a dosage schedule, for example, an initialadministration of the vaccine composition with subsequent boosteradministrations. In particular embodiments, a second dose of thecomposition is administered anywhere from two weeks to one year,preferably from about 1, about 2, about 3, about 4, about 5 to about 6months, after the initial administration. Additionally, a third dose maybe administered after the second dose and from about three months toabout two years, or even longer, preferably about 4, about 5, or about 6months, or about 7 months to about one year after the initialadministration. The third dose may be optionally administered when no orlow levels of specific immunoglobulins are detected in the serum and/orurine or mucosal secretions of the subject after the second dose. In apreferred embodiment, a second dose is administered about one monthafter the first administration and a third dose is administered aboutsix months after the first administration. In another embodiment, thesecond dose is administered about six months after the firstadministration. In another embodiment, the compositions of the inventioncan be administered as part of a combination therapy. For example,compositions of the invention can be formulated with other immunogeniccompositions, antivirals and/or antibiotics.

The dosage of the pharmaceutical composition can be determined readilyby the skilled artisan, for example, by first identifying doseseffective to elicit a prophylactic or therapeutic immune response, e.g.,by measuring the serum titer of virus specific immunoglobulins or bymeasuring the inhibitory ratio of antibodies in serum samples, or urinesamples, or mucosal secretions. The dosages can be determined fromanimal studies. A non-limiting list of animals used to study theefficacy of vaccines include the guinea pig, hamster, ferrets,chinchilla, mouse and cotton rat. Most animals are not natural hosts toinfectious agents but can still serve in studies of various aspects ofthe disease. For example, any of the above animals can be dosed with avaccine candidate, e.g. an RV VLP which comprises one or more RVglycoproteins (G proteins), to partially characterize the immuneresponse induced, and/or to determine if any neutralizing antibodieshave been produced. For example, many studies have been conducted in themouse model because mice are small size and their low cost allowsresearchers to conduct studies on a larger scale.

In addition, human clinical studies can be performed to determine thepreferred effective dose for humans by a skilled artisan. Such clinicalstudies are routine and well known in the art. The precise dose to beemployed will also depend on the route of administration. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal test systems.

As also well known in the art, the immunogenicity of a particularcomposition can be enhanced by the use of non-specific stimulators ofthe immune response, known as adjuvants. Adjuvants have been usedexperimentally to promote a generalized increase in immunity againstunknown antigens (e.g., U.S. Pat. No. 4,877,611). Immunization protocolshave used adjuvants to stimulate responses for many years, and as such,adjuvants are well known to one of ordinary skill in the art. Someadjuvants affect the way in which antigens are presented. For example,the immune response is increased when protein antigens are precipitatedby alum. Emulsification of antigens also prolongs the duration ofantigen presentation. The inclusion of any adjuvant described in Vogelet al., “A Compendium of Vaccine Adjuvants and Excipients (2^(nd)Edition),” herein incorporated by reference in its entirety for allpurposes, is envisioned within the scope of this invention.

Exemplary, adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant. Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDPcompounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, andmonophosphoryl lipid A (MPL). RIBI, which contains three componentsextracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wallskeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.MF-59, Novasomes®, MHC antigens may also be used.

In one embodiment of the invention the adjuvant is a paucilamellar lipidvesicle having about two to ten bilayers arranged in the form ofsubstantially spherical shells separated by aqueous layers surrounding alarge amorphous central cavity free of lipid bilayers. Paucilamellarlipid vesicles may act to stimulate the immune response several ways, asnon-specific stimulators, as carriers for the antigen, as carriers ofadditional adjuvants, and combinations thereof. Paucilamellar lipidvesicles act as non-specific immune stimulators when, for example, avaccine is prepared by intermixing the antigen with the preformedvesicles such that the antigen remains extracellular to the vesicles. Byencapsulating an antigen within the central cavity of the vesicle, thevesicle acts both as an immune stimulator and a carrier for the antigen.In another embodiment, the vesicles are primarily made ofnonphospholipid vesicles. In other embodiment, the vesicles areNovasomes®. Novasomes® are paucilamellar nonphospholipid vesiclesranging from about 100 nm to about 500 nm. They comprise Brij 72,cholesterol, oleic acid and squalene. Novasomes have been shown to be aneffective adjuvant for influenza antigens (see, U.S. Pat. Nos.5,629,021, 6,387,373, and 4,911,928, herein incorporated by reference intheir entireties for all purposes).

The compositions of the invention can also be formulated with “immunestimulators.” These are the body's own chemical messengers (cytokines)to increase the immune system's response. Immune stimulators include,but not limited to, various cytokines, lymphokines and chemokines withimmunostimulatory, immunopotentiating, and pro-inflammatory activities,such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13);growth factors (e.g., granulocyte-macrophage (GM)-colony stimulatingfactor (CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immunostimulatorymolecules can be administered in the same formulation as thecompositions of the invention, or can be administered separately. Eitherthe protein or an expression vector encoding the protein can beadministered to produce an immunostimulatory effect. Thus in oneembodiment, the invention comprises antigentic and vaccine formulationscomprising an adjuvant and/or an immune stimulator.

Methods of Stimulating an Immune Response

The RV VLPs which comprise one or more RV glycoproteins (G proteins) areuseful for preparing compositions that stimulate an immune response thatconfers immunity or substantial immunity to infectious agents. Theinvention encompasses a method of inducing immunity to infections or atleast one disease symptom thereof in a subject, comprising administeringat least one effective dose of an RV VLP which comprises one or more RVglycoproteins (G proteins).

In one aspect, the invention comprises a method to induce immunity to RVinfection or at least one disease symptom thereof in a subject,comprising administering at least one effective dose of an RV VLP whichcomprises one or more RV glycoproteins (G proteins). In one embodiment,the subject is a vertebrate. In another embodiment, the vertebrate is amammal. In yet another embodiment, the mammal is a human. In yet anotherembodiment, the mammal is a domestic animal. In another embodiment, themethod comprises inducing immunity to RV infection or at least onedisease symptom by administering the formulation in one dose. In anotherembodiment, the method comprises inducing immunity to RV infection or atleast one disease symptom by administering the formulation in multipledoses

Compositions of the invention can induce substantial immunity in avertebrate (e.g. a human) when administered to the vertebrate. Thesubstantial immunity results from an immune response againstcompositions of the invention that protects or ameliorates infection orat least reduces a symptom of infection in the vertebrate. In someinstances, if the vertebrate is infected, the infection will beasymptomatic. The response may not be a fully protective response. Inthis case, if the vertebrate is infected with an infectious agent, thevertebrate will experience reduced symptoms or a shorter duration ofsymptoms compared to a non-immunized vertebrate.

In another embodiment, the invention comprises a method of inducing aprotective antibody response to an infection or at least one symptomthereof in a subject, comprising administering at least one effectivedose of an RV VLP which comprises one or more RV glycoproteins (Gproteins).

As used herein, an “antibody” is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. Antibodies exist as intactimmunoglobulins or as a number of well-characterized fragments producedby digestion with various peptidases.

In one embodiment, the invention comprises a method of inducing aprotective cellular response to RV infection or at least one diseasesymptom in a subject, comprising administering at least one effectivedose of RV VLP which comprises one or more RV glycoproteins (Gproteins).

As mentioned above, the immunogenic compositions of the inventionprevent or reduce at least one symptom of RV infection in a subject.Symptoms of RV are well known in the art. They include fever, headache,and general weakness or discomfort. As the disease progresses, morespecific symptoms appear and may include insomnia, anxiety, confusion,slight or partial paralysis, excitation, hallucinations, agitation,hypersalivation (increase in saliva), difficulty swallowing, andhydrophobia (fear of water). Thus, the method of the invention comprisesthe prevention or reduction of at least one symptom associated with RVinfection. A reduction in a symptom may be determined subjectively orobjectively, e.g., self assessment by a subject, by a clinician'sassessment or by conducting an appropriate assay or measurement (e.g.body temperature), including, e.g., a quality of life assessment, aslowed progression of a RV infection or additional symptoms, a reducedseverity of a RV symptoms or a suitable assays (e.g. antibody titerand/or T-cell activation assay). The objective assessment comprises bothanimal and human assessments.

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference for all purposes.

EXAMPLES Example 1 Purification of Rabies G Particles for Animal Study

The purpose of this Example is to demonstrate how RV G virus-likeparticles were purified following expression from baculovirus vectors inSf9 insect cells.

To construct RV VLPs, the nucleic acid sequence encoding the RV Gprotein (SEQ ID NO: 2) was expressed from the baculovirus vector(pFastBac1 Rabies G) shown in FIG. 1.

Sf9 insect cells were infected at 2.5×10⁶ cell/ml with a MOI of 0.2.Cells were harvested at 69 hrs post-infection by centrifuge at 4000 gfor 15 mins. Cells were washed with 1×PBS, spun again, and frozen at−70° C.

A 23 gram cell pellet was used obtained from an approximately 2 L cellculture. The cell pellet was resuspended with 200 ml 25 mM TrisCL pH8.0, 50 mM NaCl, 0.5% NP9, 4 ug/mL leupeptin. It was stirred at roomtemperature for 1 hr, spun at 7000 g for 60 mins at 4° C., and 200 mlsupernatent was saved for chromatography.

Upon completion of Rabies G839 extraction from cell pellet, the solubleproteins were loaded onto a Fractogel EMD TMAE Hicap (M) chromatographycolumn. The specifications of the column were as follows: Columnmanufacturer: GE Healthcare; Column type: XK50f20; Resin manufacturer:EMD Chemicals; Resin type: Fractogel EMD TMAE Hicap (M); Packed columndimensions: approximately 10 cm height×5.0 cm diameter; Packed columnvolume: 200 ml; Packing flow rate: 30 ml/min; Packing buffer: 25 mM TrispH 8.0/300 mM NaCl.

The specifications of the anion exchange process were as follows:Running flow rate: 20 ml/min; Column equilibration buffer: 25 mM Tris pH8.0, 50 mM NaCl, 0.02% NP9; Eluent A: 25 mM Tris pH 8.0, 50 mM NaCl,0.02% NP9; Eluent B1: 25 mM Tris pH 8.0, 180 mM NaCl, 0.02% NP9; EluentB2: 25 mM Tris pH 8.0, 500 mM NaCl, 0.02% NP9; Eluent B3: 25 mM Tris pH8.0, 1500 mM NaCl, 0.02% NP9; Column load: 200 ml of Rabies G VLPextraction supernatant; Column wash after load: 2 CV eluent A; Columnelution: 2 CV eluent BI, 2 CV eluent B2, 2 CV eluent B3.

The major fraction collection and volumes were as follows: Flow-throughfraction: 250 mL; B1 180 mM NaCl elution: 175 ml (product); B2 500 mMNaCl elution: 100 ml; B3 1500 mM NaCl elution: 100 ml.

The 180 mM NaCl elution fraction of the TMAE column was loaded onto alentil lectin column. The specifications of the column were as follows:Column manufacturer: GE Healthcare; Column type: XKI6/20; Resinmanufacturer: GE Healthcare; Resin type: Lentil Lectin Sepharose 4B;Resin catalog #: 17-0444-01; Packed column dimensions: 2.5 cm height×1.6cm diameter; Packed column volume: approximately 5 ml; Packing flowrate: 2.5 ml/min; Packing buffer: 25 mM NaHPO₄ pH6.8, 50 mM NaCl, 0.02%NP9; Running flow rate: 2 mL/min; Column equilibration buffer: 25 mMNaHPO₄ pH6.8, 50 mM NaCl, 0.02% NP9; Eluent A: 25 mM NaHPO₄ pH6.8, 50 mMNaCl, 0.02% NP9, Eluent B: 25 mM NaHPO₄ pH6.8, 50 mM NaCl, 0.02% NP9,500 mM Methyl-alpha-Dmannopyronoside (Fisher Scientific); Column load:175 ml of Rabies G839 TMAE 180 mM NaCl elution; Column wash after load:5 CV with eluent A; Column elution: 10 CV with eluent B;

The major fraction collection and volumes were as follows: Flow-throughfraction: 180 ml; Elution fraction: 30 ml (product).

The lentil lectin elution was loaded onto a Fractogel EMD SO3—Hicap (M)chromatography column. The specifications of the column were as follows:Column manufacturer: GE Healthcare; Column type: XK16/20; Resinmanufacturer: EMD Chemicals; Resin type: Fractogel EMD SO3—Hicap (M);Packed column dimensions: 5 cm height×1.6 cm diameter; Packed columnvolume: 10 ml; Packing flow rate: 7.5 ml/min; Packing buffer: 25 mMNaHPO₄ pH 6.8, 50 mM NaCl, 0.02% NP9

The specifications of the cation exchange process were as follows:Running flow rate: 5 mL/min; Column equilibration buffer: 25 mM NaHPO₄pH 6.8, 50 mM NaCl, 0.02% NP9; Eluent A: 25 mM NaHPO₄ pH 6.8, 50 mMNaCl, 0.02% NP9; Eluent B: 25 mM NaHPO₄ pH 6.8, 300 mM NaCl, 0.02% NP9;Column load: 30 mL of Rabies G839 lectin lectin elution; Column washafter load: 3 CV with eluent A; Column elution: 4 CV step elution eluentB; Major fraction collection and volumes: Elution fraction: 9 ml (finalproduct); Filter 9 ml SO3—column 300 mM NaCl elution product with 0.2 μmfilter: Filter manufacture (0.2 μm): Corning; Filter type: 28 mm syringefilter with a 0.2 micron SFCA membrane.

Western blotting using anti-RV G rabbit sera were performed (FIG. 2).The purity of RV G particles using the above-conditions was 86%. Thetotal protein amount was 0.39 mg/ml, and the concentration of RV Gparticles was 0.33 mg/ml, with a total of 2.97 mg RV G particles from a2 L cell culture, with a yield of approximately 1.5 mg/L cell culture.Importantly, RV G particles were stable at 4° C. for at least one month(data not shown).

Example 2 Electron Microscopy for Analysis of RV G Protein Conformation

Purified RV G protein was analyzed by negative stain electron microscopy(see FIG. 3). The average molecular weight of the RV G particles with0.02% NP9 was 1.04×10⁻⁶. The protein trimers exhibited a molecularweight of 175.5 kDa and the average number of trimers in a particle was5.9. The RV G proteins aggregated in the form of micelles (rosettes).The fact that the G spikes exhibit micelle morphology under electronmicroscopy suggests that the G protein particles have the correct3-dimensional structure of a native protein.

Example 3 RV G Particles Induce High Antibody Levels in Rabbits

To test the ability of RV G particles to induce an immune response,rabbits were administered RV G particles at varying concentrations. Theresults of these experiments are illustrated in FIG. 4. RV G particleswere able to induce high levels of antibodies in rabbits.

Example 4 RV Neutralization Assay and RV Challenge Studies in Mice

To test the efficiency of a vaccine comprising RV VLPs comprising one ormore G proteins in protecting against RV infection, neutralizationassays are conducted in mice. Briefly, groups of mice are injectedintramuscularly with RV VLPs or RV VLPs+an adjuvant, such as aluminum.In addition, mice are injected with Rabipur®, a commercially availableinactivated rabies virus vaccine, which is used as a comparative vaccineagent. RV VLPs comprising one or more G proteins (i.e. RV G micelles)are generally expected to induce higher titers of neutralizingantibodies when compared with Rabipur®.

Example 5

Comparison of Anti-Rabies Titer in Balb/c Mice Injected with Either RV GParticles or Commercial Rabies Vaccine Rabipur®

The immunogenecity of the VLPs of the present invention was compared tothe commercial rabies vaccine Rabipur® in a Balb/c mouse model. RV G VLPparticles were constructed and purified as described in Example 1. TheVLPs aggregated in the form of micelles (FIG. 3).

The study included four groups (n=5 for each group):

Group I: positive control (commercial rabies vaccine Rabipur®)

Group II: RV G VLP (5 μg) Group III: RV G VLP (2 μg) Group IV: RV G VLP(1 μg)

Mice were administered the respective immunogen at 0.1 mL at days 0, 3and 7. Serum samples were taken from the mice at days 0, 4, 7, 10, 14,21, 28, 35. Serum was tested for neutralizing anti-rabies antibodies byELISA.

A summary of the study design is given in table 1 below.

TABLE 1 Study design Parameter Anti-rabies titer in serum by ELISA(neutralizing) Analytes Serum Identification Group I Group II Group IIIGroup IV Positive control Test 1 Test 2 Test 3 Number of 5 5 5 5 miceImmunogen Commercial RV G VLP RV G VLP RV G VLP rabies vaccine (5 μg) (2μg) (1 μg) Rabipur ® Schedule for 0.1 mL, I/D, Day 0, 3, 7 immunica-tion Bleeds 0, 4, 7, 10, 14, 21, 28, 35 (days) Analyte Serum forneutralizing anti-rabies virus titer

As discussed above, anti-rabies neutralizing antibodies were measured inmouse sera at the time points provided above. The titers were plotted asthe geometric mean for each measurement (FIG. 6). As FIG. 6 shows, theVLP micelles of the present invention, at each dosage, provide morerapid and higher antibody titers than the commercially availablevaccine, Rabipur®. Specifically, the RV G VLP provided a sero-protectiontiter (0.5 EU/mL) days earlier than the Rabipur® vaccine (FIG. 6, Table2).

Table 2 also shows the percent sero-protection (i.e., a neutralizingantibody titer of ≥0.5 EU/mL) for each immunization group. The tableindicates that sero-protection occurs more rapidly in animals treatedwith the RV G VLP of the present invention (at all three dosages),compared to animals administered Rabipur® vaccine.

TABLE 2 Percent sero-protection for each immunization group. %Sero-protection on day (n = 5) Identification 0 7 10 14 21 28 35Rabipur ® alone 0 0 40 60 60 60 60 (Group I) RV G VLP (5 μg) 0 0 60 100100 100 100 (Group II) RV G VLP (2 μg) 0 40 80 100 100 100 100 (GroupIII) RV G VLP (1 μg) 0 0 60 100 100 100 100

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom as modifications will be obvious to those skilled in the art.It is not an admission that any of the information provided herein isprior art or relevant to the presently claimed inventions, or that anypublication specifically or implicitly referenced is prior art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Although the application has been broken into sections to direct thereader's attention to specific embodiments, such sections should be notbe construed as a division amongst embodiments. The teachings of eachsection and the embodiments described therein are applicable to othersections.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. A purified micelle comprising one or more rabies virus (RV)glycoproteins (G proteins).
 2. The purified micelle of claim 1, whereinthe RV G protein is derived from a RV strain selected from human,canine, bat, raccoon, skunk, and fox.
 3. A virus-like particle (VLP)comprising a RV G protein.
 4. The VLP of claim 3, wherein the RV Gprotein is derived from a RV strain selected from human, canine, bat,raccoon, skunk, and fox.
 5. The VLP of claim 3, wherein the VLPcomprises a matrix (M) protein.
 6. The VLP of claim 5, wherein said Mprotein is derived from RV.
 7. The VLP of claim 5, wherein said Mprotein is M1 from an influenza virus strain.
 8. The VLP of claim 5,wherein said influenza virus strain is an avian influenza virus strain.9. The VLP of claim 8, wherein said avian influenza virus strain is anH5N1 strain.
 10. The VLP of claim 9, wherein said H5N1 strain isA/Indonesia/5/05.
 11. The VLP of claim 5, wherein said M protein isderived from a Newcastle Disease Virus (NDV) strain.
 12. The VLP ofclaim 3, wherein the VLP is expressed in a eukaryotic cell underconditions which permit the formation of VLPs.
 13. The VLP of claim 12,wherein the eukaryotic cell is selected from the group consisting ofyeast, insect, amphibian, avian, mammalian, or plant cells. 14.(canceled)
 15. An immunogenic composition comprising a VLP according toclaim
 3. 16.-17. (canceled)
 18. A kit for immunizing a human subjectagainst a viral infection comprising a purified micelle according toclaim
 1. 19. A kit for immunizing a human subject against a viralinfection comprising a VLP according to claim
 3. 20. The kit accordingto claim 18, wherein the viral infection is a RV infection. 21.-26.(canceled)
 27. An isolated nucleic acid encoding a RV G protein of SEQID NO:
 2. 28. An isolated cell comprising a nucleic acid of claim 27.29. A vector comprising the nucleic acid of claim
 27. 30.-33. (canceled)